Current research in plant molecular biology is directed toward the development of improved plant varieties through the use of genetic engineering. Historically, improved plant varieties have been developed using classical genetic techniques to identify, preserve and crossbreed plants having desired traits. However, the genetic traits available to the classical breeder are limited to those that can be identified in the particular plant species the breeder is seeking to improve.
Advances in the application of the techniques of molecular biology to plants now allow for the introduction of new traits isolated from entirely different species into the plant of interest, particularly major crop plants such as cotton, maize, sorghum, soybeans, alfalfa, tobacco, and brassicas, such as rape. Traits that have been successfully transferred include insect resistance, herbicide resistance, stress tolerance, drought resistance, and disease resistance. Present day recombinant DNA technology has made it possible to identify new genes which effect the properties of plants and of products made from plants when they are transformed into new plant species. For example, a number of insect resistant varieties of cotton are presently being grown. Crop plants resistant to the herbicides Roundup, Buctril, and Liberty Link are now available, as are tomatoes which can be left on the vine longer than normal tomatoes, making mechanical harvesting of tomatoes easier and cheaper.
A variety of techniques have been used to introduce foreign genes into plant cells. However, most of these techniques are limited to use with plant tissues that must be regenerated into whole plants and require a period of time in tissue culture. Methods of regenerating whole plants from cells or tissues include, micropropagation of apical and lateral meristems, organogenesis, and somatic embryogenesis. Transformation of apical meristems, lateral meristems and organogenesis produce chimeric plants, i.e., plants which have the gene encoding the newly introduced trait in only a few cells, which may or may not be in the gene in germline tissue. Plants regenerated through somatic embryogenesis are rarely chimeric. Somatic embryos are usually derived from a single cell.
One common method used to introduce foreign genes into plant cells is transformation with Agrobacterium, a relatively benign natural plant pathogen. Agrobacterium actively mediates transformation events--the integration of a gene providing a desired phenotypic trait--as part of the natural process it utilizes when it infects a plant cell. Methods for transferring foreign genes into plant cells and the subsequent expression of the inserted genes in plants regenerated from transformed cells are well known in the prior art. See for example, M. De Block et al., The EMBO Journal (1984) 3:1681; Horsch et al. Science (1985) 227:1229; and C. L. Kado (Crit. Rev. Plant. Sci. (1991) 10:1.
The technique known as microprojectile bombardment has been used to successfully introduce genes encoding new genetic traits into a number of crop plants, including cotton, maize, tobacco, sunflowers, soybeans and certain vegetables. See for example, U.S. Pat. No. 4,945,050, issued to Sanford; Sanford et al., Trends in Biotechnology (1988) 6:299; Sanford et al., Part. Sci. Technol. (1988) 5:27; J. J. Finer and M. D. McMullen, Plant Cell Reports (1990) 8:586-589; and Gordon-Kamm, The Plant Cell (1990) 2:603). Transformation by microprojectile bombardment is less species and genotype specific than transformation with Agrobacterium, but the frequencies of stable transformation events achieved following bombardment can be quite low, partly due to the absence of a natural mechanism for mediating the integration of a DNA molecule or gene responsible for a desired phenotypic trait into the genomic DNA of a plant. Particle gun transformation of cotton for example, has been reported to produce no more than one clonal transgenic plant per 100-500 meristems targeted for transformation. Only 0.1 to 1% of these transformants were capable of transmitting foreign DNA to progeny. See WO 92/15675. Cells treated by particle bombardment must be regenerated into whole plants, which requires labor intensive, sterile tissue culture procedures and is generally genotype dependent in most crop plants, particularly so in cotton. Similar low transformation frequencies have been reported for other plant species as well.
The inability to control the site of wounding of a plant tissue and thus the site to which the transforming agent is delivered is a significant disadvantage of microprojectile bombardment. The inability to target germline tissues accounts in part for the low transformation efficiencies achieved by microprojectile bombardment. In addition, bombardment frequently results in the delivery of more than one copy of the transforming DNA or gene into the genome of the transformed plant cell, which can cause deleterious effects to other agronomically important traits of the regenerated, transformed plant. Fragmentation of the DNA to be inserted can also occur when bombardment is used as the transformation method, resulting in transgenic plants which carry only a portion of the gene that is being inserted.
Attempts to improve the efficiency of microprojectile bombardment have been described. For example, tissues which have been bombarded are subsequently treated with an Agrobacterium carrying the gene of interest, as described in EPA 0486 233. The high velocity impact of the dense microprojectile particles has been hypothesized to generate an array of microwounds, creating an environment particularly conducive to infection by the Agrobacterium. However, these procedures provide transformed plant cells which must still be regenerated into whole plants and the fertile, stably transformed plants must be selected from the total population of regenerated plants.
Organogenesis, the development of plantlets from specific plant structures such as leaf disks or root tips, has been used to regenerate plants following transformation. However, organogenesis frequently produces plants which have originated from a group of cells, not just a single cell, and results in a chimeric plant containing both transformed and nontransformed cells. If the desired trait is to be passed on to subsequent generations of the plant, the introduced DNA must be incorporated into the genetic material of germline cells of the regenerated plant. If a mixture of transformed and nontransformed cells are involved in the regeneration of a new plant, only a portion of its cells will contain the gene encoding the transferred characteristic. The regenerated plant will be chimeric, and its germline cells may not be transformed at all. Successful transformation of plants requires that germline cells of the plant be transformed in such a way that progeny of the plant inherit the inserted gene. Otherwise, the introduced trait will be lost from progeny of the transformed plantlet.
Somatic embryogenesis, the development of embryos from somatic tissue, has been the method of choice for regenerating plants from transformed tissue. Somatic embryogenesis is superior to organogenesis in that the resulting regenerated plant is not chimeric. Somatic embryos are derived from a single cell thus all cells in the embryo contain the introduced DNA. Unfortunately, somatic embryogenesis is highly genotype dependent in most crop plants.
Methods for in planta transformation have been attempted to circumvent the time and expense of existing transformation techniques. Feldman has shown that it is feasible to vacuum infiltrate the floral meristems of small plants, or in the case of Arabidopsis, the entire plant with Agrobacterium and obtain transgenic progeny. See K. A. Feldman et al., "Agrobacterium-Mediated Transformation of Germinating Seeds of Arabidopsis thaliana: A Non-Tissue Culture Approach," Mol. Gen. Genet. (1987) 308:1-9.) Unfortunately, this technique is not feasible for large crop plants such as cotton, maize and soybeans.
Efforts have been made to develop transformation methods that deliver naked DNA to the germinating pollen tube of a plant, and subsequently to the egg cell in the floral tissue. Although expression of the inserted foreign genes has been observed, the transformants have proven to be unstable in future generations. The selection of stable, transformed plants produced by such procedures is extremely difficult due to the magnitude of the selection work involved in identifying the few transformants among the large numbers of plants that must be screened. Unlike the selection of transformed cells and tissues, which can be carried out under laboratory conditions, selection of transformed plants germinated from seed requires growth of sufficient numbers of plants in a green house or in an open field to allow identification of transformants.
Direct injection of floral tissues with a transforming agent has been attempted using ordinary syringes having needles. Direct injection with a needle results in excessive tissue damage, and provides little control of placement of the transforming agent. While direct injection with a needle places the transforming DNA within the plant, it must still be taken up by individual cells and it must be incorporated into the plant's genome. Again, since transformation frequencies of these methods are expected to be extremely low, selection of transformed plants is difficult.
Therefore, there still exists a need for a procedure that will allow the delivery of a transforming agent or DNA to germline tissues such that the agent or DNA will be incorporated directly into the DNA of the cells in these tissues, particularly into the DNA of the egg cells of the plant. A method which effectively and directly targets germline tissues would greatly improve the frequency with which the transforming agent is inserted into the genomic DNA of the germline tissues of the plant and is thus passed on to the progeny of the transformed plant.
The present invention provides an improved method for delivering transforming agents to germline tissues such that the agent or DNA will be incorporated directly into the DNA of the cells in these tissues, particularly into the DNA of the egg cells of the plant. A method which effectively and directly targets germline tissues would greatly improve the frequency with which the transforming agent is inserted into the genomic DNA of the germline tissues of the plant and is thus passed on to the progeny of the transformed plant.
The present invention further provides an improved method for delivering transforming agents to plant tissues, which overcomes deficiencies of the prior art methods by providing for the precise injection of a transforming agent, without causing excessive injury to the injected tissues. The method is particularly useful for delivering a transforming agent to developing floral tissues of a plant prior to or during seed development. The transforming agent may be a genetically engineered or recombinant Agrobacterium carrying a gene capable of conferring a desired phenotypic trait, or even a naked DNA molecule capable of conferring the desired trait. The method uses a needleless-injection device that is capable of injecting a small high pressure stream of a solution through the many cell layers of plant tissue. In one preferred embodiment of the invention the transforming agent is delivered to a plant's floral tissues, thereby facilitating delivery of a transforming agent comprising a gene of interest into germline cells of the plant. The high pressure stream provided by the injection device insures that the Agrobacterium culture or the DNA solution penetrates the many cell layers of the plant floral tissue without causing massive tissue damage, such as that caused by direct injection with a syringe having a needle or by particle bombardment. One of skill in the art of plant molecular biology will understand that the method of the present invention can also be adapted for transformation of plant cells and tissues, including embryonic tissue culture cells, meristematic tissues and plant callus, which can be regenerated into whole plants. They will also recognize that the method can be adapted for introducing DNAs conferring phenotypic traits into plant tissues and cells to be used in transient transformation assays and in other assays used in plant research.